Abstract: Pelvic organ prolapse (POP) is the downward descent of the pelvic organs due to weakening of the pelvic support structures, such as the uterosacral ligament (USL). It is a common disorder that negatively impacts the quality of life of millions of women. POP is a multifactorial condition with contributions from genetic and environmental factors, and both have been shown to equally contribute in liability. Aging, menopause, vaginal delivery, and increasing BMI are well established risk factors associated with POP. The mechanisms by which these factors influence gene expression in the pelvic support ligaments are poorly understood, and lack of such knowledge has severely limited progress in prevention and treatment of POP. Epigenetics may be a mechanism to explain this concept and is defined as heritable changes in gene expression that occur without alterations in DNA sequence. Methylation of cytosines in CpG dinucleotides is a well-defined epigenetic modification and is associated with alterations in gene expression. To date, no epigenome-wide studies have been performed in POP research. We have preliminary data showing altered DNA methylation in the HOXA11 gene in the USLs in women with POP. This homeobox gene is important for USL homeostasis. The goal of this proposal is to determine the extent to which epigenetic mechanisms contribute to the etiology of POP in women in an epigenome-wide study. We hypothesize that risk factors for POP modulate the methylation state and transcriptional activity of critical genes that affect homeostasis of the USLs. In specific aim 1, we will measure epigenome-wide methylation patterns in USL biopsy specimens from postmenopausal women with normal pelvic support and with POP that are matched in age, vaginal parity, and BMI. We will prioritize target genes differing in methylation profiles using the Encyclopedia of DNA Elements (ENCODE) and Roadmap Epigenomics (RE) data and will validate the methylated sites of interest. We will determine if USLs in women with POP contain altered DNA methylation profiles within the variant genes that have been identified in epigenome-wide methylation analysis studies or within the HOXA11 gene. In specific aim 2, we will determine the influence of POP risk factors (age, vaginal parity, and BMI) on significant epigenetic marks that differ in women with POP vs. the CTL group and confirm the expression of target genes with novel methylated loci using quantitative PCR to elucidate their functional significance. Identifying unique epigenetic marks and linking them to risk factors for POP will help our understanding of the etiology and pathogenesis of POP, and lead to more effective, targeted and personalized approaches to disease prevention and treatment.